Stimulatory Effect of Indolic Hormone on As2O3 Cytotoxicity in Breast Cancer Cells: NF-κB-dependent Mechanism of Action of Melatonin

The advent of combination therapy unprecedentedly shifted the paradigm of cancer treatment by reconstructing the conventional protocols. By identifying the anti-tumoral activity for different natural products, recent interest has focused on inventing the combined- modality strategies to increase the cure rates of cancer, while reducing the toxic side effects of current intensive regimens. To evaluate whether melatonin, indolic hormone produced mainly by the pineal gland, could enhance the pro-apoptotic effect of arsenic trioxide (As2O3) in breast cancer, MCF-7 cells were treated with As2O3-plus- melatonin and then the survival, proliferative rate, caspase-3 activity, and mRNA expression level of anti- apoptosis target genes of NF-κB were investigated. Our results delineated that exposure of MCF-7 cells to As2O3 not only reduced the survival of the cells, but also induced a caspsase-3-dependent apoptotic cell death. Noteworthy, an enhanced induction of apoptosis was found using As2O3 in combination with melatonin. Moreover, RQ-PCR analysis revealed that the enhanced cytotoxic effect of As2O3 in the presence of melatonin is mediated, at least partly, through suppressing the expression of NF-κB anti-apoptotic target genes such as MCL-1, BCL-2, survivin, XIAP, and c-IAP1 in breast cancer cells. The resulting data showed that As2O3, either alone or in combination with melatonin, exerted significant cytotoxic effect against MCF-7 cells. However, further investigations are needed to provide valuable clues for expediting this combination as a therapeutic strategy for breast cancer.

he therapeutic approaches in breast cancer, as the most common malignancy in womankind in term of morbidity and mortality (1), still remain an unsolvable dilemma; therefore, the entrance of the novel anti-tumoral agents in combination with chemotherapy or radiotherapy turns to be groundbreaking for more efficient treatment.
During the last decade, identifying the potent anticancerous property of arsenic trioxide (As2O3) revitalizes the popularity of this agent as an effective chemotherapeutic drug not only for hematologic malignancies but also for solid tumors (2). An overwhelming number of studies imply that As2O3 exerts its variegated apoptotic effects through various mechanisms including modulation of the intracellular glutathione redox system (3), induction of mitotic arrest (4), DNA damage and inhibition of DNA repair (5). Despite its prodigious anti-tumoral activities, the clinical use of this agent became restricted due to the toxic side effects of long-term intake of high doses of As2O3. A recent renaissance of As2O3 following the novel disclosure indicating that As2O3 is an ideal agent to be used in combination therapy, has revived the interest in this ancient drug and has constructed its future direction into clinical investigations.
Melatonin, the master biologic clock neurohormone, is basically considered as the main regulator of circadian rhythm and sleep quality (6).
Thus far, a plethora of biological actions have been reported for melatonin; however, recent investigations concerning its inhibitory impact on the development, promotion, and progression of various types of cancers, in particular, breast cancer have attracted tremendous attention (7). In the last decades, several experimental studies have outlined that melatonin not only possess a chemo-preventive property (8), but also exerts a pro-apoptotic activity in cancer cells mostly through generation of intercellular ROS (9,10). For the nonce, multiple published reports have discussed about the intensifying effect of melatonin in combination with different chemotherapeutic drugs, especially DNA damaging agents such as cis-platin (11), cyclophosphamide (12), and doxorubicin (13).
Based on the convergence mechanisms between melatonin and As2O3 (14,15), it was tempting to evaluate whether melatonin could enhance the proapoptotic effect of As2O3 in MCF-7 breast cancer cells.

Trypan blue exclusion assay
Breast cancer-derived MCF-7 cells were seeded at the density of 450 × 10 5 cells and were incubated with various concentrations of As2O3 and melatonin, either alone or in combined modality.
After 48 h, drug treated-cells were trypsinized, centrifuged and the cell pellets were re-suspended T in phosphate-buffered saline (PBS) in combination with 0.4 % trypan blue (Invitrogen, New Zealand).
By using a Neubauer hemocytometer, the number of viable cells was counted and afterwards the percentage of viable cells was assessed using the following formula: viability (%)= viable cell count/total cell count×100.

MTT assay
Microculture tetrazolium assay (MTT) was applied to explore the impeding effect of As2O3, Finally, the cells were exposed to 100 μl of substrate tetramethyl-benzidine (TMB) for 3 min at room temperature and the reaction product was quantified by measuring the absorbance at 450 nm in an ELISA reader.

Median-effect analysis of drug combinations
To evaluate whether there was a synergistic effect between As2O3 and melatonin, we computed the combination index (CI) using the method developed by Chou and Talalay (16). Values of CI and DRI after 48 h treatment of MCF-7 cells are summarized in Table 2.